Linda B Kidd, Gernot A Schabbauer, James P Luyendyk, Todd D...

JPET #138891

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Title Page

Insulin Activation of the PI3K/Akt Pathway Reduces LPS-induced Inflammation in

Mice

Linda B Kidd, Gernot A Schabbauer, James P Luyendyk, Todd D Holscher, Rachel E

Tilley, Michael Tencati, and Nigel Mackman

The Department of Immunology, The Scripps Research Institute, La Jolla CA

JPET Fast Forward. Published on April 29, 2008 as DOI:10.1124/jpet.108.138891

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 29, 2008 as DOI: 10.1124/jpet.108.138891

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Running Title Page

Running title: Insulin inhibits LPS-induced inflammation in mice

Correspondence should be addressed to Nigel Mackman, Ph.D at the Division of

Hematology/Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Tel 919 843 3961, Fax 919 966 7639, email [email protected].

Number of text pages: 25

Number of tables: 1

Number of figures: 4

Number of references: 40

Number of words in

Abstract: 203

Introduction: 459

Discussion: 948

Non-standard Abbreviations: phosphatidylinositol 3-kinase (PI3K), protein kinase B

(Akt), keratinocyte chemoattractant (KC), tumor necrosis factor α (TNFα), interleukin 6

(IL-6), mitogen activated protein kinase phosphatase (MKP-1), glycogen synthase kinase

3 beta (GSK- 3β), TNFα converting enzyme (TACE)

Section Affiliation: Inflammation, Immunopharmacology and Asthma


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Abstract

Insulin is used to control pro-inflammatory hyperglycemia in critically ill patients.

However, recent studies suggest that insulin induced hypoglycemia may negate its

beneficial effects in these patients. Importantly, recent evidence indicates that insulin has

anti-inflammatory effects that are independent of controlling hyperglycemia. To date, the

mechanism by which insulin directly reduces inflammation has not been elucidated. It is

well established that insulin activates PI3K/Akt signaling in many cell types. We and

others have shown that this pathway negatively regulates LPS-induced signaling and pro-

inflammatory cytokine production in monocytic cells. We hypothesized that insulin

inhibits inflammation during endotoxemia by activation of the phosphatidylinositol 3-

kinase (PI3K)/protein kinase B (Akt) pathway. We used a non-hyperglycemic mouse

model of endotoxemia to determine the effect of continuous administration of a low dose

of human insulin on inflammation and survival. Importantly, insulin treatment induced

phosphorylation of Akt in muscle and adipose tissues but did not exacerbate LPS induced

hypoglycemia. Insulin decreased plasma levels of IL-6, TNFα, JE and KC, and decreased

mortality. The PI3K inhibitor wortmannin abolished the insulin-mediated activation of

Akt and the reduction of chemokine and interleukin-6 levels. We conclude that insulin

can reduce LPS-induced inflammation in mice in a PI3K/Akt dependent manner without

affecting blood glucose levels.


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Introduction

During Gram-negative sepsis, lipopolysaccharide (LPS) induces the expression of

pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα) and interleukin 6

(IL-6), and chemokines, such as JE and keratinocyte chemoattractant (KC) (Casey, et al.,

1993). However, overreaction of the pro-inflammatory response to infection and injury

can contribute to the development of sepsis, septic shock, multiple organ failure, and

death. Hyperglycemia and insulin resistance followed by hypoglycemia and

hypoinsulinemia can also occur during sepsis as a consequence of the metabolic effects

of stress hormone and cytokine production (Van den Berghe, 2004;Marik and Raghavan,

2004;van Waardenburg, et al., 2006;Maitra, et al., 2000). Despite considerable progress

in our understanding of the pathologic pathways that contribute to sepsis and septic

shock, pharmacologic interventions are currently limited to insulin and activated protein

C (Shapiro, et al., 2006). Insulin is administered to septic patients with hyperglycemia in

order to normalize glucose levels (Russell, 2006;Shapiro, et al., 2006). Reducing glucose

levels with insulin therapy is associated with decreased inflammation and endothelial cell

damage (Van den Berghe, et al., 2001;Van den Berghe, 2004;Van den Berghe, et al.,

2006;Marik and Raghavan, 2004;Langouche, et al., 2005). Recently however, it has been

shown that insulin-induced hypoglycemia may counteract the beneficial effects of

aggressive insulin therapy in patients with severe sepsis (Brunkhorst 2008).

Currently, insulin is not initiated in normoglycemic septic patients or to correct

hypoinsulinemia during sepsis (Mitchell, et al., 2006). However, results from animal

studies indicate that insulin may have direct anti-inflammatory effects. In non-

hyperglycemic animal models of endotoxemia, continuous infusion of insulin while


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maintaining blood glucose in the normal range with dextrose, or administering a bolus

injection of insulin results in decreased inflammation and morbidity (Jeschke, et al.,

2004;Jeschke, et al., 2005;Brix-Christensen, et al., 2004). However, the mechanism by

which insulin reduces inflammation in the absence of hyperglycemia is unknown. One

possibility is that insulin reduces inflammation by activating anti-inflammatory signaling

pathways, such as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)

pathway. It is well established that insulin activates PI3K/Akt signaling in many cell

types (Taniguchi, et al., 2006). Importantly, we and others have shown that this pathway

negatively regulates LPS-induced signaling and pro-inflammatory cytokine production in

monocytic cells (Guha and Mackman, 2002;Martin, et al., 2005;Liew, et al.,

2005;Schabbauer, et al., 2004). Furthermore, activation of PI3K enhanced survival

whereas inhibition of PI3K reduced survival of endotoxemic mice (Schabbauer, et al.,

2004;Williams, et al., 2004). Taken together, these studies suggest that the protective

effects of insulin in endotoxemia models may be mediated by activation of the PI3K/Akt

pathway.

In this study, we used a pharmacological inhibitor of PI3K to test the hypothesis

that exogenous insulin decreases inflammation in endotoxemic mice by activating the

PI3K/Akt pathway, and that this antiinflammatory effect is independent of its affect on

blood glucose levels.


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Methods

Mice. All studies were approved by The Scripps Research Institute Animal Care

and Use Committee and comply with National Institutes of Health guidelines. C57BL/6J

male mice of 6-16 weeks of age were used for all experiments.

Mouse endotoxemia model and insulin administration. We used a mouse

model of endotoxemia consisting of an intraperitoneal (i.p.) injection of LPS

(Escherichia coli serotype O111:B4; Sigma Chemical Company, St Louis, Mo). Doses

of 5 mg/kg or 10 mg/kg were used in experiments. These doses corresponded to the LD50

for two different lots of LPS. Accordingly, LPS produced similar levels of cytokine

expression and lethality in each experiment. Human insulin (Humulin® 70/30; Eli Lilly,

Indianapolis, IN) was administered to mice either as a bolus i.p. injection or via Alzet®

micro-osmotic pumps (Model 1003D; Durect Corp, Cupertino, CA) that were implanted

subcutaneously 16 hours before administration of LPS. These pumps deliver 1 µL/hour

for 72 hours. Human insulin was used to allow us to measure low levels of exogenous

insulin and to distinguish it from endogenous mouse insulin in the endotoxemic mice.

Furthermore, human insulin has been used previously in studies examining the effect of

acute insulin treatment in endotoxemic rodents (Jeschke et al., 2004). To inhibit PI3K

activity, mice were given wortmannin (Sigma Chemical Company) at a dose of 0.06

mg/kg or its vehicle (Ringer’s solution containing 10% (v/v) dimethyl sulfoxide) 3 times

by retro-orbital injection at -90, +90, and +360 min relative to LPS administration. Blood

samples were collected from the retro-orbital sinus (final conc. 0.32%) at various times

after LPS administration (0-24 hours). Plasma was collected and stored at -80°C until

analysis.


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Measurement of glucose, insulin, and cytokine levels. Glucose levels in the

plasma were determined using a Glucometer Elite XL (Bayer Corporation, Elkhart, IN).

Human insulin levels in the plasma were determined using a commercial ELISA that

detects only human insulin (Mercodia 10-1132-01; Uppsala, Sweden). Total insulin

(mouse + human) levels in the plasma were determined using a different commercial

ELISA kit (Mercodia 10-1150-01). The levels of TNFα, IL-6, JE, and KC in plasma

were determined using commercial ELISA kits (R&D Systems, Minneapolis, MN).

Western Blotting. Muscle and epididymal adipose tissue were collected and

quick frozen in liquid nitrogen and stored at -80°C until processed. Tissues were

homogenized in 1.5mL of a buffer 10mM HEPES, 10mM KCl, 300mM sucrose, 1.5mM

MgCl2, 0.5mM DTT, 0.5mM PMSF, 1/2 tablet and complete EDTA-free protease

inhibitor cocktail (Roche, Indianapolis, IN), spun at 550xg for 2 min at 4°C and

supernatants frozen. Protein concentration was measured using a colorimetric assay

according to manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). Proteins

were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) and then transferred to Millipore polyvinylidene flouride membranes (Millipore,

Bedford, MA). Levels of phosphorylated (Ser 473) and non-phosphorylated Akt were

detected using primary rabbit antibodies (Cell Signaling, Beverly, MA) and a secondary

antibody conjugated to horseradish peroxidase. Antibodies were developed using a

chemiluminescence reagent assay (Super Signal West Femto; Pierce, Rockford, IL)

according to manufacturer’s instructions, and exposed to radiographic film (Kodak,

Rochester, NY).


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Statistical Analysis. Data were analyzed by one-way analysis of variance with

Tukey’s test for multiple comparisons or by Student’s t-test. Non-normal data were

analyzed by the Mann-Whitney rank sum or the Kruskal-Wallis One Way Analysis of

Variance on ranks and Dunn’s method of multiple comparisons. Survival data were

analyzed using the log-rank test. The criterion for significance for all studies was p<0.05.


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Results

Effect of exogenous insulin on plasma insulin and glucose levels in untreated

and LPS-treated mice. Two different bolus doses of human insulin were used to

determine a dose that did not affect plasma glucose levels in fasted male mice. A 1 U/kg

dose of human insulin (Humulin® 70/30) administered i.p. to mice significantly reduced

plasma glucose levels, whereas a 0.1 U/kg dose of insulin had no effect (Table 1).

Therefore, the 0.1 U/kg dose of human insulin was selected as the dose for all further

studies. We used micro-osmotic infusion pumps to continuously infuse insulin

throughout the study period. These pumps delivered insulin at a rate of 2.5 mU/hr/mouse

(approximately 0.1 U/kg/hr) subcutaneously (SQ) for 72 hours. Low levels of human

insulin were detected in insulin-treated mice but not in saline-treated controls using an

ELISA specific for human insulin (Fig. 1A). Human insulin levels were maintained in

insulin-treated mice at a constant level for the first 8 hours of endotoxemia. Total insulin

(mouse + human) was measured using a second ELISA. There was no significant

difference in total insulin levels between insulin-treated and saline treated mice during

endotoxemia (Fig. 1B). Importantly, insulin-treatment did not exacerbate LPS-induced

hypoglycemia (Fig 1C).

Exogenous insulin decreases inflammation and mortality in endotoxemic

mice. Insulin treatment significantly reduced plasma levels of TNFα, IL-6, JE, and KC

and improved the survival of endotoxemic mice compared with mice treated with saline

(Fig. 2A-F). Interestingly, we observed a negative correlation between plasma levels of

human insulin and IL-6 (data not shown).


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Exogenous insulin activates Akt in insulin-senstive tissues in endotoxemic

mice. We hypothesized that the protective effects of insulin during endotoxemia are

mediated by activation of PI3K/Akt. We chose to analyze the activation of Akt in muscle

and adipose tissue because glucose transport in theses tissues is regulated by insulin and

they express inflammatory mediators during endotoxemia (Chang, et al., 2004;Bultinck,

et al., 2006;Brix-Christensen, et al., 2005). Importantly, Akt phosphorylation was

increased in the muscle and maintained in adipose tissue of insulin-treated endotoxemic

mice (Fig. 3A-B).

Effect of wortmannin on the anti-inflammatory activity of insulin. To

investigate the role of the PI3K/Akt pathway in the anti-inflammatory effects of insulin

during endotoxemia, we treated mice with the PI3K inhibitor wortmannin. The dose of

wortmannin used in these experiments did not affect plasma glucose levels (data not

shown) but abolished insulin-dependent Akt phosphorylation in the muscle of

endotoxemic mice (Fig. 3C). Wortmannin also abolished the insulin-dependent decrease

in IL-6, JE and KC levels at 8 hours (Fig. 4A-C). Interestingly, wortmannin increased

KC expression during endotoxemia (Fig. 4B). However, wortmannin did not inhibit the

insulin-dependent reduction in TNFα levels at 1.5 hours (Fig. 4D).


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Discussion

In this study, we analyzed the mechanism by which insulin reduces LPS-induced

inflammation in mice. We used a non-hyperglycemic model of endotoxemia, and

continuously infused insulin at a dose and rate that did not worsen LPS-induced

hypoglycemia. We showed that a low dose of human insulin decreased levels of TNFα,

IL-6, JE, and KC, and decreased mortality in endotoxemic mice without exacerbating

hypoglycemia. We then used a pharmacologic approach to analyze the role of PI3K/Akt

signaling in the anti-inflammatory effects of insulin. The low dose of human insulin used

in this study enhanced phosphorylation of Akt in muscle and adipose tissue during the

hypoglycemic, hypoinsulinemic phase of endotoxemia. Wortmannin abolished the

insulin-induced phosphoryation of Akt in these mice indicating inhibition of PI3K.

Importantly, inhibition of insulin-mediated activation of PI3K/Akt with wortmannin

reversed the insulin-dependent reduction in LPS-induced IL-6, JE and KC expression.

These data indicate that insulin activation of the PI3K/Akt pathway inhibits the

production of these cytokines in endotoxemic mice. However, because wortmannin

inhibits other cell signaling pathways, we cannot exclude the possibility that additional

mechanisms also contribute to the anti-inflammatory effects of insulin in these mice

(Ding et al, 1995).

It is interesting to note that insulin-dependent reduction in TNFα production was

not reversed by wortmannin. This observation may, in part, relate to the complexity of

TNFα release in endotoxemia. For example, previous studies have shown that the

cleavage of the pro-form of TNFα from the membrane by TACE contributes significantly

to plasma TNFα levels in endotoxemic mice (Zhang et al, 2004). Further studies are


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required to determine whether insulin-mediated activation of PI3K plays a role in

reducing TNFα levels in endotoxemic mice.

Other studies indirectly support our observation that activation of PI3K/Akt

signaling mediates many of the anti-inflammatory effects of insulin during endotoxemia.

For instance, insulin has been shown to increase the expression of MAPK phosphatase

(MKP-1) in a PI3K-dependent manner (Takehara, et al., 2000;Desbois-Mouthon, et al.,

2000), and this phosphatase negatively regulates IL-6 and TNFα production in

endotoxemic mice (Chi, et al., 2006). Furthermore, a recent study showed that insulin

and a GSK3β inhibitor had similar anti-inflammatory effects in a rat model of sepsis

(Dugo, et al., 2006). Importantly, Akt-dependent phosphorylation decreases the activity

of GSK3β (Guha and Mackman, 2002;Martin, et al., 2005). These studies, together with

the findings reported here support the notion that activation of the PI3K/Akt pathway

mediates the protective effects of insulin during endotoxemia.

Both increased levels of IL-6 and hypoinsulinemia have been associated with

increased morbidity and decreased survival during endotoxemia and sepsis (Casey, et al.,

1993;van Waardenburg, et al., 2006;Ciancio, et al., 1991). In this study, endogenous

insulin levels decreased dramatically at a time when IL-6 levels were maximal. In insulin

treated mice, the maximum amount of human insulin in plasma 8 hours after LPS was 8.9

mU/L. However, as a consequence of LPS-induced hypoinsulinemia, this small amount

of insulin constituted a large portion of total insulin levels 8 hours after the administration

of LPS. Notably, we observed a strong negative correlation between human insulin

levels and plasma IL-6 levels in insulin-treated mice. These data suggest that small


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amounts of insulin can significantly impact the inflammatory response without affecting

plasma glucose levels.

Glucose transport into muscle and adipose is partially dependent on insulin-

mediated activation of the PI3K/Akt pathway. Insulin mediated effects on glucose

metabolism in the liver are also dependent on activation of this pathway. The dose of

insulin used in this study increased levels of phosphorylated Akt in muscle and adipose

tissue during endotoxemia. Interestingly, we did not observe increased levels of

phosphorylated Akt in the liver (data not shown). Therefore, tissue-specific differences

in the activation of the PI3K/Akt pathway may explain how a low dose of insulin can

impact the inflammatory response without affecting glucose metabolism.

The cellular target(s) mediating the protective effects of insulin in this model

remain to be determined. Insulin-dependent tissues, such as skeletal muscle and adipose

tissue, contribute to IL-6 expression during endotoxemia (Bultinck, et al., 2006;Brix-

Christensen, et al., 2005), suggesting that the anti-inflammatory effects of insulin on IL-6

production during endotoxemia may be the result of direct activation of PI3K in these

tissues. Hematopoietic cells, such as monocytes and macrophages, have been shown to

be the major source of cytokine production during endotoxemia (Michalek, et al., 1980).

Insulin may directly activate PI3K in monocytes and thereby reduce inflammatory

cytokine production in response to LPS in vivo. Insulin signaling has not been studied

extensively in macrophages and mononuclear cells. Although they do not require insulin

for glucose transport, these cells express insulin receptors and insulin increases glucose

transport (Welham, et al., 1997;Estrada, et al., 1994;Daneman, et al., 1992). A recent

study showed that high concentrations of insulin (104µM) decreased TNFα and IL-1β


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production and inhibited apoptosis in LPS treated THP-1 cells in vitro (Leffler, et al.,

2007). The anti-apoptotic effects of insulin were mediated by activation of PI3K.

However, this study did not determine whether the anti-inflammatory effects of insulin

were PI3K dependent.

Apoptosis of cells, particularly endothelial cells, may contribute to inflammation

and organ damage during endotoxemia (Power, et al., 2002;Bannerman and Goldblum,

2003). Interestingly, insulin inhibits apoptosis in a variety of cell types in a PI3K/Akt-

dependent manner and may reduce endothelial apoptosis in endotoxemic mice (Conejo

and Lorenzo, 2001;Hermann, et al., 2000;Leffler, et al., 2007). Further studies are

required to determine the cellular targets of insulin during endotoxemia.

In summary, we demonstrated that continuous administration of small amounts of

insulin decreased inflammation in endotoxemic mice in a PI3K-dependent manner

without affecting blood glucose levels. These findings may ultimately have important

implications regarding the use of insulin for treating normoglycemic, hypoinsulinemic

patients with sepsis.


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Acknowledgements

We would like to thank Cheryl Johnson for preparing the manuscript and Dr. T.

Combs for critical reading of the manuscript.


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Footnotes

Authors Kidd, Schabbauer and Luyendyk contributed equally to the work

This work is supported by National Institutes of Health Grant HL48872 (N.M.), National

Institutes of Health NSRA F32 HL085983 (J.P.L.), Austrian Science Fund FWFP19850

(G.S.), and National Institutes of Health NRSA T32 5 P32 HL007195-30 (L.K.).


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Legends for Figures

Figure 1: Measurement of human insulin, total insulin, and glucose in normal and

endotoxemic mice. Plasma levels of (A) human insulin (n=12 per group), (B) total

insulin (mouse + human) (n=14 mice per group), and (C) plasma glucose (n=20 mice per

group) were measured by ELISA (insulin) or glucometer (glucose) at various times (0-8

hours) after LPS. Results are displayed as mean ± SEM.

Figure 2: Administration of human insulin decreases inflammation and mortality in

endotoxemic mice. Human insulin (2.5 mU/uL/hr) or saline (1 µL/hr) was administered

continuously to mice using a subcutaneously implanted pump. Plasma levels of (A)

TNFα (n=6-10 mice per group), (B) IL-6 (n=10-12 mice per group), (C) JE (n=5-6), and

(D) KC (n=5-7) were measured by ELISA prior to and 1, 8 and 24 hours after LPS.

Results are shown as mean ± SEM. (E) Survival was evaluated for 4 days. Results are

presented as a Kaplan-Meier plot (n=10 mice/group). * indicates p<0.05.

Figure 3: Effect of human insulin and wortmannin on Akt phosphorylation in

muscle and adipose tissue in endotoxemic mice. Human insulin (2.5 mU/µLhr) or

saline (1 µL/hr) was administered continuously to mice using a subcutaneously implanted

pump before treating the mice with LPS with or without wortmannin. The left

quadriceps muscle and adipose tissue were harvested 8 hours after administration of LPS.


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Non-fasted, untreated mice served as controls. Levels of phosphorylated Akt and total

Akt were detected by western blotting in muscle (A and C) and adipose tissue (B).

Representative data from 1-3 independent mice in each group is shown.

Figure 4. Effect of wortmannin on the anti-inflammatory activity of insulin in

endotoxemic mice. Human insulin (2.5 mU/µL/hr) or saline (1µL/hr) was continuously

administered to mice using subcutaneously implanted pumps. Mice were treated with

LPS and/or wortmannin. Plasma levels of IL-6 (A), JE (B) and KC (C) (8 hours after

LPS) and TNFα (D) (1.5 hours) were measured in the 4 groups. Results are displayed as

the mean ± SEM (n=5-12 mice per group). * indicates p<0.05. Wort=wortmannin.


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Table 1: Establishment of a dose of insulin that does not affect blood glucose

Insulin Dose 0.1 U/kg 1.0 U/kg

Time after insulin (min) 0 20 60 120 0 20 60 120

Plasma glucose mg/dL

(mean ± SEM)

n=3-6 mice/group

99.8

±

10.8

83.0

±

13.3

94.3

±

6.9

95.3

±

7.1

99.8

±

10.8

69.7

±

8.0

54.3*

±

9.0

79.0

±

14.5

* significantly different from time zero, p<0.05


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